The present invention relates to a method for recording an image in transmission electron microscopy (hereinafter referred to as TEM or “electron microscope”). Electron microscopes use a focused beam of electrons instead of light to image a specimen and gain information as to its structure or composition. Transmission electron microscopes pass image-forming rays through the specimen being observed. Contrast or diffracted beam images can be used to analyze the specimen or sample. Conventionally, recording of an electron microscope image has been affected with a photographic film.
More particularly, it concerns such a method of recording an image on a film element adaptable to presently existing TEM instrumentation designs without modification of the machinery and, furthermore, makes possible TEM exposure followed by immediate processing of film.
TEM instruments are capable of providing an image of a specimen with a magnification factor of up to one million times and are used extensively in such fields as medicine, biology, chemistry, metallurgy, material science, and other industrial applications for visible observation of such magnified images. Electron microscopy is also used for measuring or inspection of semiconductors or other products or components of products. Although the magnified electron image may be observed directly when focused on a fluorescent screen or by using other forms of electronic imaging devices, the resolution of detail in such directly observable images is much lower than the resolving capacity of photographic emulsions. For this reason, as well as for providing permanent records of TEM magnified images of specimens, TEM instruments are conventionally equipped with photographic film exposing systems to enable visual observation of high-resolution detail in the magnified specimen image. Moreover, final analyses of a given specimen is usually delayed until one or more photographs of the TEM image are available for observation.
TEM instruments typically comprise high power electron beam generating and focusing components, and the space or chamber in which the electrons are transmitted must be evacuated to 10−7 atmospheric pressure or more in order to avoid electron scattering by collision with molecules of air or with molecules of other substances in a gaseous phase. In this latter respect, it is to be noted that all normally liquid and even some normally solid substances may vaporize under the magnitude of vacuums developed in the electron chamber of TEM instruments. In some more advanced instruments, the film for recording the image is held at a lower vacuum (less negative pressure), for example, 10−5 rather than 10−7 mm Hg, by the use of a differential aperture positioned between the column and the “camera chamber,” the latter holding the film for exposure and optionally a detector and viewing screen. In any case, whether at a lower vacuum or not, the film and film handling accessories of a TEM photographic system are typically presented in an evacuated camera chamber that receives the electron beam for exposing the film. Moreover, the film is passed into and out of the camera chamber, and each TEM instrument involves costly vacuum sealing mechanisms predicated in substantial part on the physical format of film unit assemblies employed and on the configuration of film containers or boxes to be used in a TEM instrument of a given design. Hence, modification of photographic components in presently existing TEM equipment is impractical and, moreover, design changes in photographic apparatus supplied by manufacturers of TEM instruments are restricted to accommodation of respective TEM instrument designs.
The skilled artisan will appreciate that in the present use of TEM instrumentation, the attainment of a high resolution photograph of a specimen is a very tedious and time consuming procedure by which the benefits of specimen analysis are significantly delayed. This is particularly true in the field of pathological analysis of tissue removed by surgery or in similar fields where it would be desirable to have the benefit of a TEM photograph available within a short period of time. Also, in the material sciences where electron microscopy is used for quality control or production problem solving a fast turn around time is desirable.
In TEM, conventional films employ silver-halide emulsions similar to those used based on light exposure. In films exposed with light, electron hole pairs are generated and silver specks, clusters, or latent images are generated by actinic radiation. In TEM, the electron beam interacts with the silver halide grains directly to generate the silver specks, clusters, or latent images. Although electron beams are technically not electromagnetic radiation, the net result is essentially the same. Because photographic technology involving exposure to such light or radiation is so common, such terms as “radiation,” “light-sensitive” and “photography” and “light-sensitive” are often applied, respectively, to “electron beams,” “electron-sensitive” and imaging based on electron exposure. Thus, such terms will be used interchangeably herein as will be appreciated by the skilled artisan.
In conventional photography, films containing light-sensitive silver-halide grains are employed in a number of image recording devices including but not limited to x-ray and electron-imaging elements. Upon exposure, the film produces a latent image that is only revealed after suitable processing. These film elements have historically been processed by treating the exposed film with at least a developing solution having a developing agent that acts to form an image in cooperation with other components in the film.
It is always desirable to limit the amount of solvent or processing chemicals used in the processing of silver-halide films. The traditional photographic processing scheme for black-and-white film involves development, fixing and washing, each step typically involving immersion in a tank holding the necessary chemical solution. By the use of photothermographic film, it is possible to eliminate processing solutions altogether, or alternatively, to minimize the amount of processing solutions and the complex chemicals contained therein. A photothermographic (PTG) film by definition is a film that requires energy, typically heat, to effectuate development. A dry photothermographic film requires only heat. A solution-minimized photothermographic film may require a small amount of aqueous alkaline solution to effectuate development, for example, an amount required to swell the film without excess solution. Development is the process whereby silver ion is reduced to metallic silver and in a color system, a dye is created in an image-wise fashion. In many photothermographic films, the silver is typically retained in the coating after thermal development.
In photothermographic films employing what is referred to as “dry physical development,” a photosensitive catalyst (also an electron-sensitive catalyst) is generally a photographic-type photosensitive silver halide that is considered to be in catalytic proximity to a non-photosensitive (or non-electron-sensitive) source of reducible silver ions. Catalytic proximity requires intimate physical association of these two components either prior to or during the thermal image development process so that when silver atoms, (Ago)n, also known as silver specks, clusters, nuclei, or latent image, are generated by irradiation or light exposure of the photosensitive silver halide, those silver atoms are able to catalyze the reduction of the reducible silver ions within a catalytic sphere of influence around the silver atoms (Klosterboer, Neblette's Eighth Edition: Imaging Processes and Materials, Sturge, Walworth & Shepp (eds.), Van Nostrand-Reinhold, New York, Chapter 9, pages 279–291, 1989). It has long been understood that silver atoms act as a catalyst for the reduction of silver ions, and that the photosensitive silver halide can be placed in catalytic proximity with the non-photosensitive source of reducible silver ions in a number of different ways (see, for example, Research Disclosure, June 1978, item 17029. Research Disclosure is a publication of Kenneth Mason Publications Ltd., Dudley House, 12 North Street, Emsworth, Hampshire PO10 7DQ England and also available from Emsworth Design Inc., 147 West 24th Street, New York, N.Y. 10011). Research Disclosure, September 1996, Number 389, Item 38957 is hereafter referred to as “Research Disclosure I”.
The non-photo-sensitive source of reducible silver ions is typically a material that contains reducible silver ions and preferably a silver salt of an organic compound.
Photothermographic (PTG) media employing dry physical development are formulated with one or more light sensitive imaging layers on a light transmitting or reflecting support. Each imaging layer typically has at least one light-sensitive silver-halide emulsion, a reducible non-light-sensitive silver salt, a developer or developer precursor, and optionally a coupler to form dye. Other components may include accelerators, toners, binders, and antifoggants known in the trade as well as components used in conventional solution-processed silver-halide photographic media. Such PTG media are similarly applicable to electron microscopy using a silver-halide emulsion, in which electrons replace light as the source of exposure.
When exposed to light or electrons (the “exposing energy”) and then heated at temperatures ranging from 100 to 200° C. for 5 to 60 seconds, photothermographic media develop densities varying with exposure. The density versus log exposure curve (H&D curve) is commonly used in the trade to compare parameters such as speed and contrast. A typical procedure for generating the H&D curve entails making a contact exposure through a step tablet image. The steps modulate the intensity of the incident exposing energy such as light, usually in 0.10 to 0.30 log exposure increments. Another method entails exposing pixel-wise using a laser, CRT or LED source in which the exposure intensity is modulated electronically.
The measured reflection or transmission density of each step on the photographic media for light exposure is typically plotted against relative or absolute log exposure to produce what is known in the industry as the “H&D curve.” H&D curves typically have two plateaus corresponding to the maximum density (Dmax) and minimum density (Dmin) where the slope of the H&D curve approaches or equals zero; that is, a change in exposure produces little or no change in measured density. Gamma refers to the slope of the H&D curve usually at some fixed density position. Point gamma refers to the change in density between two adjacent exposure positions in a plot of the H&D values. The mid-scale density refers to the density midway between Dmax and Dmin plateaus, or (Dmax-Dmin)/2. The corresponding exposure is designated the mid-scale exposure. In contrast, electron microscopy, rather than the use of a step tablet, an H&D curve can be obtained by making multiple exposures varying time and/or intensity. The H&D curve or response from electron exposures has an exponential shape. Whereas in light exposure, the gamma provides a measurement of contrast and is constant for a given film and processing condition, electron exposure has a constant change in gamma because of the exponential shape. Consequently, an increase of contrast is obtained by increasing density which can be obtained by exposure or changes in the processing conditions.
As used herein with respect to the present invention, the term “negative-working” refers to a photographic silver-halide emulsion that develops more density with increasing exposure up to a maximum density when an imagewise-exposed gelatin coating of the emulsion is processed using a solution-development process and concomitant materials in accordance with the well-known and conventional D-76 standard. The corresponding H&D curve has a positive (but changing) slope in the mid-scale density range when density is plotted against increasing relative log exposure. The unexposed areas develop to Dmin. The image produced in this way is referred to as a “negative image.” It is to be understood that the term “negative-working emulsion” as used herein is synonymous with “potentially negative-working emulsion” and refers to an inherent capability of the emulsion that may or may not be realized in practice.
A “positive-working” photographic silver-halide emulsion, as used herein with respect to the present invention, responds to exposure by developing less density with increasing exposure down to the a lower limit (Dmin) when an imagewise-exposed gelatin coating of the emulsion is processed using a solution-development process and materials in accordance to the well-known D-19 standard. In this case, the H&D curve has a negative (but changing) slope in the mid-scale density region when density is plotted against increasing relative log exposure. The unexposed areas develop to a maximum density. The image produced in this way is referred to as a “positive image.”
Materials, including solution developers, qualifying for commercially acceptable use in a D-19 standard process include Kodak's trademarked products designed for such a process. See G. Haist, “Modern Photographic Processing, Vol 1”, John Wiley & Sons, Chapter 7, p 340 (1979) for the preparation of D-19 developer and other related developer formulas, hereby incorporated by reference. D-19 developer, therefore, includes any or all materials designated for and commercially used, with commercially satisfactory results in a D-19 process. Preferably, the D-19 developer is a Kodak product or one that is substantially equivalent in practice.
In a positive-working or negative-working emulsion, the developed density can comprise either silver, or if the imaging layer also contains a dye-forming coupler to react with oxidized developer, silver plus dye.
In the case of conventional solution-processed photographic media, as compared to dry or apparently dry thermally developed photothermographic media, positive images can be obtained from negative-working emulsions using combinations of multiple exposures and/or multiple development steps. See G. Haist, cited above, for details on black-and-white and color reversal-development processes, in which the following patents are cited: U.S. Pat. No. 2,005,837, U.S. Pat. No. 2,126,516, U.S. Pat. No. 2,184,013, U.S. Pat. No. 2,699,515, U.S. Pat. No. 3,361,564, U.S. Pat. No. 3,367,778, U.S. Pat. No. 3,455,235, U.S. Pat. No. 3,501,310, U.S. Pat. No. 3,519,428, U.S. Pat. No. 3,560,213, U.S. Pat. No. 3,579,345, U.S. Pat. No. 3,650,758, U.S. Pat. No. 3,655,390, BR 44248, BR 1151782, BR 1155404, BR 1186711, BR 1201792, CA 872180, and CA 872181.
For example, photobleach emulsions can be used in conventional solution-developed silver-halide photographic media to produce positive images. These emulsions are prepared with desensitizing dyes and chemical fogging agents. An exposure destroys preformed surface fog centers rendering the grains undevelopable. The unexposed grains develop to form a positive image. G. Haist reviews this topic in Modern Photographic Processing, Vol 2, Chapter 7, John Wiley & Sons, (copyright 1979).
Commonly assigned copending application Ser. No. 10/460,142, Filed Jun. 12, 2003, relates to a positive-working silver-halide photothermographic film that can be exposed by various forms of energy including ultraviolet and infrared regions of the spectrum as well as electron beam and beta radiation, gamma ray, x-ray, alpha particle, neutron radiation and other forms of corpuscular wave-like radiant energy. The film can be use for high speed black and white film, including consumer camera film, x-ray film, dental film, and dosimeters.
Positive-working photographic silver-halide emulsions are not generally used for imaging in electron microscopy. There are no known positive-working photothermographic silver-halide emulsions that are sensitive to focused electron-beams.
Also, a significant problem with photothermographic elements has been the difficulty obtaining high photographic speeds. Organic solvents may degrade photographic efficiency. Methods of chemical and spectral sensitizations in organic solvents are less effective than in water for similar reasons. Gelatin coatings, on the other hand, are more difficult to thermally develop due to the physical properties of the gelatin when it is heated. Lower developed density and photographic speed generally result from the higher glass transition temperature of gelatin and generally slower rates of diffusion of developer components in the strong hydrogen bonding polypeptide matrix. Gelatin coatings also require dispersing the incorporated water-insoluble developer components, which causes them to react generally more sluggishly under thermal processing conditions compared to organic solvent coatings in which developer components are dissolved in the coating solvent.
The prior art describes photothermographic systems that produce negative images that are nearly equal in speed to those obtained with solution development. In contrast, the present invention can produce direct-positive photographic speeds that are significantly greater than speeds obtained by solution or thermal development of same-size negative-working silver-halide emulsions.